Papilloma virus capsomere vaccine formulations and methods of use

ABSTRACT

Vaccine formulations comprising viral capsomeres are disclosed along with methods for their production. Therapeutic and prophylactic methods of use for the vaccine formulations are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/358,285 filed Feb. 21, 2006, now U.S. Pat. No. 7,371,391which is a divisional application of U.S. patent application Ser. No.10/042,526, which was filed on Jan. 8, 2002, now abandoned which is acontinuation application of U.S. patent application Ser. No. 09/632,286,which was filed on Aug. 3, 2000, now abandoned which is a continuationof U.S. patent application Ser. No. 08/944,368, which was filed on Oct.6, 1997 now U.S Pat. No. 6,228,368.

FIELD OF THE INVENTION

The present invention relates to vaccine formulations comprisingpapilloma virus proteins, either as fusion proteins, truncated proteins,or truncated fusion proteins. The invention further embraces methods forproducing capsomeres of the formulations, as well as prophylactic andtherapeutic methods for their use.

BACKGROUND

Infections with certain high-risk strains of genital papilloma virusesin humans (HPV)—for example, HPV 16, 18, or 45—are believed to be themain risk factor for the formation of malignant tumors of the anogenitaltract. Of the possible malignancies, cervical carcinoma is by far themost frequent; according to an estimate by the World Health Organization(WHO), almost 500,000 new cases of the disease occur annually. Becauseof the frequency with which this pathology occurs, the connectionbetween HPV infection and cervical carcinoma has been extensivelyexamined, leading to numerous generalizations.

For example, precursor lesions of cervical intraepithelial neoplasia(CIN) are known to be caused by papilloma virus infections [Crum, NewEng. J. Med. 310:880-883 (1984)]. DNA from the genomes of certain HPVtypes, including for example, strains 16, 18, 33, 35, and 45, have beendetected in more than 95% of tumor biopsies from patients with thisdisorder, as well as in primary cell lines cultured from the tumors.Approximately 50 to 70% of the biopsied CIN tumor cells have been foundto include DNA derived only from HPV 16.

The protein products of the HPV 16 and HPV 18 early genes E6 and E7 havebeen detected in cervical carcinoma cell lines as well as in humankeratinocytes transformed in vitro [Wettstein, et al., in PAPILLOMAVIRUSES AND HUMAN CANCER, Pfister (Ed.), CRC Press: Boca Raton, Fla.1990 pp 155-179] and a significant percentage of patients with cervicalcarcinoma have anti-E6 or anti-E7 antibodies. The E6 and E7 proteinshave been shown to participate in induction of cellular DNA synthesis inhuman cells, transformation of human keratinocytes and other cell types,and tumor formation in transgenic mice [Arbelt, et al., J. Virol.,68:4358-4364 (1994); Auewarakul, et al., Mol. Cell. Biol. 14:8250-8258(1994); Barbosa, et al., J. Virol. 65:292-298 (1991); Kaur, et al., J.Gen. Virol. 70:1261-1266 (1989); Schlegel, et al., EMBO J. 7:3181-3187(1988)]. The constitutive expression of the E6/E7 proteins appears to benecessary to maintain the transformed condition of HPV-positive tumors.

Despite the capacity of some HPV strains to induce neoplastic phenotypesin vivo and in vitro, still other HPV types cause benign genital wartssuch as condylomata acuminata and are only rarely associated withmalignant tumors [Ikenberg, In Gross, et al., (eds.) GENITALPAPILLOMAVIRUS INFECTIONS, Springer Verlag: Berlin, pp., 87-112]. Lowrisk strains of this type include, for example, HPV 6 and 11.

Most often, genital papilloma viruses are transmitted between humansduring intercourse which in many instances leads to persistent infectionin the anogenital mucous membrane. While this observation suggests thateither the primary infection induces an inadequate immune response orthat the virus has developed the ability to avoid immune surveillance,other observations suggest that the immune system is active duringprimary manifestation as well as during malignant progression ofpapilloma virus infections [Altmann et al. in VIRUSES AND CANCER, Minsonet al., (eds.) Cambridge University Press, (1994) pp. 71-80].

For example, the clinical manifestation of primary infection by rabbitand bovine papilloma virus can be prevented by vaccination with wartextracts or viral structural proteins [Altmann, et al., supra; Campo,Curr. Top. In Microbiol and Immunol. 186:255-266 (1994); Yindle andFrazer, Curr. Top. In Microbiol. and Immunol. 186; 217-253 (1994)].Rodents previously vaccinated with vaccinia recombinants encoding HPV 16early proteins E6 or E7, or with synthetic E6 or E7 peptides, aresimilarly protected from tumor formation after inoculation of HPV 16transformed autologous cells [Altman, et al., supra; Campo, et al.,supra; Yindle and Frazer, et al. supra]. Regression of warts can beinduced by the transfer of lymphocytes from regressor animals followinginfection by animal papilloma viruses. Finally, in immunosuppressedpatients, such as, for example, recipients of organ transplants orindividuals infected with HIV, the incidence of genital warts, CIN, andanogenital cancer is elevated.

To date, no HPV vaccinations have been described which comprise humanpapilloma virus late L1 protein in the form of capsomeres which aresuitable both for prophylactic and therapeutic purposes. Since the L1protein is not present in malignant genital lesions, vaccination with L1protein does not have any therapeutic potential for these patients.Construction of chimeric proteins, comprising amino acid residues fromL1 protein and, for example E6 or E7 protein, which give rise tochimeric capsomeres, combines prophylactic and therapeutic functions ofa vaccine. A method for high level production of chimeric capsomereswould therefore be particularly desirable, in view of the possibleadvantages offered by such a vaccine for prophylactic and therapeuticintervention.

Thus there exists a need in the art to provide vaccine formulationswhich can prevent or treat HPV infection. Methods to produce vaccineformulations which overcome problems known in the art to be associatedwith recombinant HPV protein expression and purification wouldmanifestly be useful to treat the population of individuals alreadyinfected with HPV as well as useful to immunize the population ofindividuals susceptible to HPV infection.

SUMMARY OF THE INVENTION

The present invention provides therapeutic and prophylactic vaccineformulations comprising chimeric human papilloma capsomeres. Theinvention also provides therapeutic methods for treating patientsinfected with an HPV as well as prophylactic methods for preventing HPVinfection in a susceptible individual. Methods for production andpurification of capsomeres and proteins of the invention are alsocontemplated.

In one aspect of the invention, prophylactic vaccinations for preventionof HPV infection are considered which incorporate the structuralproteins L1 and L2 of the papilloma virus. Development of a vaccine ofthis type faces significant obstacles because papilloma viruses cannotbe propagated to adequate titers in cell cultures or other experimentalsystems to provide the viral proteins in sufficient quantity foreconomical vaccine production. Moreover, recombinant methodologies toexpress the proteins are not always straightforward and often results inlow protein yield. Recently, virus-like particles (VLPs), similar inmake up to viral capsid structures, have been described which are formedin Sf-9 insect cells upon expression of the viral proteins L1 and L2 (orL1 on its own) using recombinant vaccinia or baculovirus. Purificationof the VLPs can be achieved very simply by means of centrifugation inCsCl or sucrose gradients [Kimbauer, et al., Proc. Natl. Acad. Sci.(USA), 99:12180-12814 (1992); Kirnbaurer, et al., J. Virol. 67:6929-6936(1994); Proso, et al., J. Virol. 6714:1936-1944 (1992); Sasagawa, etal., Virology 2016:126-195 (1995); Volpers, et al, J. Virol.69:3258-3264 (1995); Zhou, et al., J. Gen. Virol. 74:762-769 (1993);Zhou, et al., Virology 185:251-257 (1991)]. WO 93/02184 describes amethod in which papilloma virus-like particles (VLPs) are used fordiagnostic applications or as a vaccine against infections caused by thepapilloma virus. WO 94/00152 describes recombinant production of L1protein which mimics the conformational neutralizing epitope on humanand animal papilloma virions.

In another aspect of the invention, therapeutic vaccinations areprovided to relieve complications of, for example, cervical carcinoma orprecursor lesions resulting from papilloma virus infection, and thusrepresent an alternative to prophylactic intervention. Vaccinations ofthis type may comprise early papilloma virus proteins, principally E6 orE7, which are expressed in the persistently infected cells. It isassumed that, following administration of a vaccination of this type,cytotoxic T-cells might be activated against persistently infected cellsin genital lesions. The target population for therapeutic interventionis patients with HPV-associated pre-malignant or malignant genitallesions. PCT patent application WO 93/20844 discloses that the earlyprotein E7 and antigenic fragments thereof of the papilloma virus fromHPV or BPV is therapeutically effective in the regression, but not inthe prevention, of papilloma virus tumors in mammals. While early HPVproteins have been produced by recombinant expression in E. coli orsuitable eukaryotic cell types, purification of the recombinant proteinshas proven difficult due to inherent low solubility and complexpurification procedures which generally require a combination of steps,including ion exchange chromatography, gel filtration and affinitychromatography.

According to the present invention, vaccine formulations comprisingpapilloma virus capsomeres are provided which comprise either: (i) afirst protein that is an intact viral protein expressed as a fusionprotein comprised in part of amino acid residues from a second protein;(ii) a truncated viral protein; (iii) a truncated viral proteinexpressed as a fusion protein comprised in part of amino acid residuesfrom a second protein, or (iv) some combination of the three types ofproteins. According to the invention, vaccine formulations are providedcomprising capsomeres of bovine papilloma virus (BPV) and humanpapilloma virus. Preferred bovine virus capsomeres comprise protein frombovine papilloma virus type I. Preferred human virus capsomeres compriseproteins from any one of human papilloma virus strains HPV6, HPV11,HPV16, HPV18, HPV33, HPV35, and HPV45. The most preferred vaccineformulations comprise capsomeres comprising proteins from HPV16.

In one aspect, capsomere vaccine formulations of the invention comprisea first intact viral protein expressed as a fusion protein withadditional amino acid residues from a second protein. Preferred intactviral proteins are the structural papilloma viral proteins L1 and L2.Capsomeres comprised of intact viral protein fusions may be producedusing the L1 and L2 proteins together or the L1 protein alone. Preferredcapsomeres are made up entirely of L1 fusion proteins, the amino acidsequence of which is set out in SEQ ID NO: 2 and encoded by thepolynucleotide sequence of SEQ ID NO: 1. Amino acids of the secondprotein can be derived from numerous sources (including amino acidresidues from the first protein) as long as the addition of the secondprotein amino acid residues to the first protein permits formation ofcapsomeres. Preferably, addition of the second protein amino acidresidues inhibits the ability of the intact viral protein to formvirus-like particle structures; most preferably, the second proteinamino acid residues promote capsomere formation. In one embodiment ofthe invention, the second protein may be any human tumor antigen, viralantigen, or bacterial antigen which is important in stimulating animmune response in neoplastic or infectious disease states. In apreferred embodiment, the second protein is also a papilloma virusprotein. It also preferred that the second protein be the expressionproduct of papilloma virus early gene. It is also preferred, however,that the second protein be selected from group of E1, E2, E3, E4, E5,E6, and E7—early gene products encoded in the genome of papilloma virusstrains HVP6, HPV11, HPV18, HPV33, HPV35, or HPV 45. It is mostpreferred that the second protein be encoded by the HPV16 E7 gene, theopen reading frame of which is set out in SEQ ID NO: 3. Capsomeresassembled from fusion protein subunits are referred to herein aschimeric capsomeres. In one embodiment, the vaccine formulation of theinvention is comprised of chimeric capsomeres wherein L1 protein aminoacid residues make up approximately 50 to 99% of the total fusionprotein amino acid residues. In another embodiment, L1 amino acidresidues make up approximately 60 to 90% of the total fusion proteinamino acid residues; in a particularly preferred embodiment, L1 aminoacids comprise approximately 80% of the fusion protein amino acidresidues.

In another aspect of the invention, capsomere vaccine formulations areprovided that are comprised of truncated viral proteins having adeletion of one or more amino acid residues necessary for formation of avirus-like particle. It is preferred that the amino acid deletion notinhibit formation of capsomeres by the truncated protein, and it is mostpreferred that the deletion favor capsomere formation. Preferred vaccineformulations of this type include capsomeres comprised of truncated L1with or without L2 viral proteins. Particularly preferred capsomeres arecomprised of truncated L1 proteins. Truncated proteins contemplated bythe invention include those having one or more amino acid residuesdeleted from the carboxy terminus of the protein, or one or more aminoacid residues deleted from the amino terminus of the protein, or one ormore amino acid residues deleted from an internal region (i.e., not fromeither terminus) of the protein. Preferred capsomere vaccineformulations are comprised of proteins truncated at the carboxyterminus. In formulations including L1 protein derived from HPV16, it ispreferred that from 1 to 34 carboxy terminal amino acid residues aredeleted. Relatively shorter deletions are also contemplated which offerthe advantage of minor modification of the antigenic properties of theL1 proteins and the capsomeres formed thereof. It is most preferred,however, that 34 amino acid residues be deleted from the L1 sequence,corresponding to amino acids 472 to 505 in HPV16 set out in SEQ ID NO:2, and encoded by the polynucleotide sequence corresponding tonucleotides 1414 to 1516 in the human HPV16 L1 coding sequence set outin SEQ ID NO: 1.

When a capsomere vaccine formulation is made up of proteins bearing aninternal deletion, it is preferred that the deleted amino acid sequencecomprise the nuclear localization region of the protein. In the L1protein of HPV 16, the nuclear localization signal is found from aboutamino acid residue 499 to about amino acid residue 505. Followingexpression of L1 proteins wherein the NLS has been deleted, assembly ofcapsomere structures occurs in the cytoplasm of the host cell.Consequently, purification of the capsomeres is possible from thecytoplasm instead of from the nucleus where intact L1 proteins assembleinto capsomeres. Capsomeres which result from assembly of truncatedproteins wherein additional amino acid sequences do not replace thedeleted protein sequences are necessarily not chimeric in nature.

In still another aspect of the invention, capsomere vaccine formulationsare provided comprising truncated viral protein expressed as a fusionprotein adjacent amino acid residues from a second protein. Preferredtruncated viral proteins of the invention are the structural papillomaviral proteins L1 and L2. Capsomeres comprised of truncated viralprotein fusions may be produced using L1 and L2 protein componentstogether or L1 protein alone. Preferred capsomeres are those comprisedof L1 protein amino acid residues. Truncated viral protein components ofthe fusion proteins include those having one or more amino acid residuesdeleted from the carboxy terminus of the protein, or one or more aminoacid residues deleted from the amino terminus of the protein, or one ormore amino acid residues deleted from an internal region (i.e., not fromeither terminus) of the protein. Preferred capsomere vaccineformulations are comprised of proteins truncated at the carboxyterminus. In those formulations including L1 protein derived from HPV16,it is preferred that from 1 to 34 carboxy terminal amino acid residuesare deleted. Relatively shorter deletions are also contemplated thatoffer the advantage of minor modification of the antigenic properties ofthe L1 protein component of the fusion protein and the capsomeres formedthereof. It is most preferred, however, that 34 amino acid residues bedeleted from the L1 sequence, corresponding to amino acids 472 to 505 inHPV16 set out in SEQ ID NO: 2, and encoded by the polynucleotidesequence corresponding to nucleotides 1414 to 1516 in the human HPV16 L1coding sequence set out in SEQ ID NO: 1. When the vaccine formulation iscomprised of capsomeres made up of proteins bearing an internaldeletion, it is preferred that the deleted amino acid sequence comprisethe nuclear localization region, or sequence, of the protein.

Amino acids of the second protein can be derived from numerous sourcesas long as the addition of the second protein amino acid residues to thefirst protein permits formation of capsomeres. Preferably, addition ofthe second protein amino acid residues promotes or favors capsomereformation. Amino acid residues of the second protein can be derived fromnumerous sources, including amino acid residues from the first protein.In a preferred embodiment, the second protein is also a papilloma virusprotein. It also preferred that the second protein be the expressionproduct of papilloma virus early gene. It is most preferred, however,that the second protein be selected from group of early gene productsencoding by papilloma virus E1, E2, E3, E4, E5, E6, and E7 genes. In oneembodiment, the vaccine formulation of the invention is comprised ofchimeric capsomeres wherein L1 protein amino acid residues make upapproximately 50 to 99% of the total fusion protein amino acid residues.In another embodiment, L1 amino acid residues make up approximately 60to 90% of the total fusion protein amino acid residues; in aparticularly preferred embodiment, L1 amino acids comprise approximately80% of the fusion protein amino acid residues.

In a preferred embodiment of the invention, proteins of the vaccineformulations are produced by recombinant methodologies, but informulations comprising intact viral protein, the proteins may beisolated from natural sources. Intact proteins isolated from naturalsources may be modified in vitro to include additional amino acidresidues to provide a fusion protein of the invention using covalentmodification techniques well known and routinely practiced in the art.Similarly, in formulations comprising truncated viral proteins, theproteins may be isolated from natural sources as intact proteins andhydrolyzed in vitro using chemical hydrolysis or enzymatic digestionwith any of a number of site-specific or general proteases, thetruncated protein subsequently modified to include additional amino acidresides as described above to provide a truncated fusion protein of theinvention.

In producing capsomeres, recombinant molecular biology techniques can beutilized to produce DNA encoding either the desired intact protein, thetruncated protein, or the truncated fusion protein. Recombinantmethodologies required to produce a DNA encoding a desired protein arewell known and routinely practiced in the art. Laboratory manuals, forexample Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Press: Cold Spring Harbor, N.Y. (1989) andAusebel et al., (eds.), PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, Inc. (1994-1997), describe in detail techniques necessary to carryout the required DNA manipulations. For large-scale production ofchimeric capsomeres, protein expression can be carried out using eitherviral or eukaryotic vectors. Preferable vectors include any of the wellknown prokaryotic expression vectors, recombinant baculoviruses, COScell specific vectors, vaccinia recombinants, or yeast-specificexpression constructs. When recombinant proteins are used to providecapsomeres of the invention, the proteins may first be isolated from thehost cell of its expression and thereafter incubated under conditionswhich permit self-assembly to provide capsomeres. Alternatively, theproteins may be expressed under conditions wherein capsomeres are formedin the host cell.

The invention also contemplates processes for producing capsomeres ofthe vaccine formulations. In one method, L1 proteins are expressed fromDNA encoding six additional histidines at the carboxy terminus of the L1protein coding sequence. L1 proteins expressed with additionalhistidines (His L1 proteins) are most preferably expressed in E. coliand the His L1 proteins can be purified using nickel affinitychromatography. His L1 proteins in cell lysate are suspended in adenaturation buffer, for example, 6 M guanidine hydrochloride or abuffer of equivalent denaturing capacity, and then subjected to nickelchromatography. Protein eluted from the nickel chromatography step isrenatured, for example in 150 mM NaCl, 1 mM CaCl₂, 0.01% Triton-X 100,10 mM HEPES (N-2-hydroxyethyl piperazine-N′-2 ethane sulfonic acid), pH7.4. According to a preferred method of the invention, assembly ofcapsomeres takes place after dialysis of the purified proteins,preferably after dialysis against 150 mM NaCl, 25 mM Ca²⁺, 10% DMSO(dimethyl sulfoxide), 0.1% Triton-X 100, 10 mM Tris[tris-(hydroxymethyl) aminomethane]acetic acid with a pH value of 5.0.

Formation of capsomeres can be monitored by electron microscopy, and, ininstances wherein capsomeres are comprised of fusion proteins, thepresence of various protein components in the assembled capsomere can beconfirmed by Western blot analysis using specific antisera.

According to the present invention, methods are provided for therapeutictreatment of individuals infected with HPV comprising the step ofadministering to a patient in need thereof an amount of a vaccineformulation of the invention effective to reduce the level of HPVinfection. The invention also provide methods for prophylactic treatmentof individuals susceptible to HPV infection comprising the step ofadministering to an individual susceptible to HPV infection an amount ofa vaccine formulation of the invention effective to prevent HPVinfection. While infected individuals can be easily identified usingstandard diagnostic techniques, susceptible individuals may beidentified, for example, as those engaged in sexual relations with aninfected individual. However, due to the high frequency of HPVinfection, all sexually active persons are susceptible to papillomavirus infection.

Administration of a vaccine formulation can include one or moreadditional components such as pharmaceutically acceptable carriers,diluents, adjuvants, and/or buffers. Vaccines may be administered at asingle time or at multiple times. Vaccine formulation of the inventionmay be delivered by various routes including, for example, oral,intravenous, intramuscular, nasal, rectal, transdermal, vaginal,subcutaneous, and intraperitoneal administration.

Vaccine formulations of the invention offer numerous advantages whencompared to conventional vaccine preparations. As part of a therapeuticvaccination, capsomeres can promote elimination of persistently infectedcells in, for example, patients with CIN or cervical carcinoma.Additionally, therapeutic vaccinations of this type can also serve aprophylactic purpose in protecting patients with CIN lesions fromre-infection. As an additional advantage, capsomeres can escapeneutralization by pre-existing anticapsid antibodies and thereby posseslonger circulating half-life as compared to chimeric virus-likeparticles.

Vaccine formulations comprising chimeric capsomeres can provide theadditional advantage of increased antigenicity of both proteincomponents of the fusion protein from which the capsomere is formed. Forexample, in a VLP, protein components of the underlying capsomere may beburied in the overall structure as a result of internalized positioningwithin the VLP itself. Similarly, epitopes of the protein components maybe sterically obstructed as a result of capsomere-to-capsomere contact,and therefore unaccessible for eliciting an immune response. Preliminaryresults using L1/E7 fusion proteins to produce VLPs support thisposition in that no antibody response was detected against the E7component. This observation is consistent with previous results whichindicate that the carboxy terminal region of L1 forms inter-pentamericarm structures that allow assembly of capsomeres into capsids [Garcia,et al., J. Virol. 71: 2988-2995 (1997)]. Presumably in a chimericcapsomere structure, both protein components of the fusion proteinsubstructure are accessible to evoke an immune response. Capsomerevaccines would therefore offer the additional advantage of increasedantigenicity against any protein component, including, for example,neutralizing epitopes from other virus proteins, expressed as a fusionwith L1 amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is illustrated by the following examples. Example1 describes construction of expression vectors to produce fusion, orchimeric, viral proteins. Example 2 relates to generation of recombinantbaculoviruses for expression of viral proteins. Example 3 addressespurification of capsomeres. Example 4 describes an immunization protocolfor production of antisera and monoclonal antibodies. Example 5 providesa peptide ELISA to quantitate capsomere formation. Example 6 describesan antigen capture ELISA to quantitate capsomere formation. Example 7provides a hemagglutinin assay to assay for the induction ofneutralizing antibodies.

EXAMPLE 1 Construction of Chimeric L1 Genes

DNA encoding the HPV 16 L1 open reading frame was excised from plasmid16-114/k-L1/L2-pSynxtVI⁻ [Kimbauer et al., J. Virol. 67:6929-6936(1994)] using BglII and the resulting fragment subcloned into pUC19 (NewEngland Biolabs, Beverly, Mass.) previously linearized at the uniqueBamHI restriction site. Two basic expression constructs were firstgenerated to permit subsequent insertion of DNA to allow fusion proteinexpression. One construct encoded HPV 16 L1 Δ 310 having a nine aminoacid deletion; the deleted region was known to show low level homologywith all other papilloma virus L1 proteins. The second construct, HPV 16L1 Δ C, encoded a protein having a 34 amino acid deletion of the carboxyterminal L1 residues. Other constructs include an EcoRV restriction siteat the position of the deletion for facilitated insertion of DNAencoding other protein sequences. Addition of the EcoRV site encodes twonon-L1 protein amino acids, aspartate and isoleucine.

A. Generation of an HPV 16 L1 Δ 310 expression construct

Two primers (SEQ ID NOs: 5 and 6) were designed to amplify the pUC19vector and the complete HPV 16 L1 coding sequence, except nucleotides916 through 942 in SEQ ID NO: 1. Primers were synthesized to alsointroduce a unique EcoRV restriction site (underlined in SEQ ID NOs: 5and 6) at the termini of the amplification product.

CCCCGATATCGCCTTTAATGTATAAATCGTCTGG SEQ ID NO: 5CCCCGATATCTCAAATTATTTTCCTACACCTAGTG SEQ ID NO: 6The resulting PCR product was digested with EcoRV to providecomplementary ends and the digestion product circularized by ligation.Ligated DNA was transformed into E. coli using standard techniques andplasmids from resulting colonies were screened for the presence of anEcoRV restriction site. One clone designated HPV 16 L1 Δ 310 wasidentified as having the appropriate twenty-seven nucleotide deletionand this construct was used to insert DNA fragments encoding other HPV16 proteins at the EcoRV site as discussed below.B. Generation of an HPV 16 L1 Δ C expression constructs

Two primers (SEQ ID NOs: 7 and 8) were designed complementary to the HPV16 L1 open reading frame such that the primers abutted each other topermit amplification in reverse directions on the template DNAcomprising HPV 16 L1-encoding sequences in pUC19 described above.

SEQ ID NO: 7 AAAGATATCTTGTAGTAAAAATTTGCGTCCTAAAGGAAAC SEQ ID NO: 8AAAGATATCTAATCTACCTCTACAACTGCTAAACGCAAAAAACGEach primer introduced an EcoRV restriction site at the terminus of theamplification product. In the downstream primer (SEQ ID NO: 8), theEcoRV site was followed by a TAA translational stop codon positionedsuch that the amplification product, upon ligation of the EcoRV ends tocircularize, would include deletion of the 34 carboxy terminal L1 aminoacids. PCR was performed to amplify the partial L1 open reading frameand the complete vector. The amplification product was cleaved withEcoRV, circularized with T4 DNA ligase, and transformed into E. coli DH5α cells. Plasmids from viable clones were analyzed for the presence ofan EcoRV site which would linearize the plasmid. One positive constructdesignated pUCHPV16L1 Δ C was identified and used to insert DNA fromother HPV 16 proteins utilizing the EcoRV site.C. Insertion of DNA Fragments into HPV 16 L1 Δ 310 and HPV16L1 Δ CDNA fragments of HPV 16 E7 encoding amino acids 1-50, 1-60, 1-98, 25-75,40-98, 50-98 in SEQ ID NO: 4 were amplified using primers thatintroduced terminal 5′ EcoRV restriction sites in order to facilitateinsertion of the fragment into either HPV 16 L1 Δ310 and HPV16L1 Δ Cmodified sequence. In the various amplification reactions, primer E7.1(SEQ ID NO: 9) was used in combination with primer E7.2 (SEQ ID NO: 10)to generate a DNA fragment encoding E7 amino acids 1-50; with primerE7.3 (SEQ ID NO: 11) generate a DNA fragment encoding E7 amino acids1-60; or with primer E7.4 (SEQ ID NO: 12) generate a DNA fragmentencoding E7 amino acids 1-98. In other amplification reactions, primerpairs E7.5 (SEQ ID NO: 13) and E7.6 (SEQ ID NO: 14) were used to amplifya DNA fragment encoding E7 amino acids 25-75; E7.7 (SEQ ID NO: 15) andE7.4 (SEQ ID NO: 12) were used to amplify a DNA fragment encoding E7amino acids 40-98; and E7.8 (SEQ ID NO: 16) and E7.4 (SEQ ID NO: 12)were used to amplify a DNA fragment encoding E7 amino acids 50-98.

Primer E7.1 SEQ ID NO: 9 AAAAGATATCATGCATGGAGATACACCTACATTGC Primer E7.2SEQ ID NO: 10 TTTTGATATCGGCTCTGTCCGGTTCTGCTTGTCC Primer E7.3 SEQ ID NO:11 TTTTGATATCCTTGCAACAAAAGGTTACAATATTGTAATGGGCC Primer E7.4 SEQ ID NO:12 AAAAGATATCTGGTTTCTGAGAACAGATGGGGCAC Primer E7.5 SEQ ID NO: 13TTTTGATATCGATTATGAGCAATTAAATGACAGCTCAG Primer E7.6 SEQ ID NO: 14TTTTGATATCGTCTACGTGTGTGCTTTGTACGCAC Primer E7.7 SEQ ID NO: 15TTTATCGATATCGGTCCAGCTGGACAAGCAGAACCGGAC Primer E7.8 SEQ ID NO: 16TTTTGATATCGATGCCCATTACAATATTGTAACCTTTTG

Similarly, nucleotides from DNA encoding the influenza matrix protein(SEQ ID NO: 17) was amplified using the primer pair set out in SEQ IDNOs: 19 and 20. Both primers introduced an EcoRV restriction site in theamplification product.

SEQ ID NO: 19 TTTTGATATCGATATGGAATGGCTAAAGACAAGACCAATC SEQ ID NO: 20TTTTGATATCGTTGTTTGGATCCCCATTCCCATTG

PCR products from each amplification reaction were cleaved with EcoRVand inserted into the EcoRV site of either the HPV 16 L1 Δ 310 andHPV16L1 Δ C sequences previously linearized with the same enzyme. Inorder to determine the orientation of inserts in plasmids encoding E7amino acids 25-75 and 50-98 and plasmid including influenza matrixprotein, ClaI digestion was employed, taking advantage of a restrictionsite overlapping the newly created EcoRV restriction site (GATATCGAT)and included in the upstream primer. For the three expression constructsincluding the initiating methionine of HPV16 E7, insert orientation wasdetermined utilizing a Ns/I restriction site within the E7 codingregion.

Once expression constructs having appropriate inserts were identified,the protein coding region for both L1 and inserted amino acids wasexcised as a unit using restriction enzymes XhaI and SmaI and theisolated DNA ligated into plasmid pVL1393 (Invitrogen) to generaterecombinant baculoviruses.

D. Elimination of EcoRV Restriction Sites in Expression Constructs

The HPV 16 L1 Δ C sequence includes DNA from the EcoRV site that resultsin translation of amino acids not normally found in wild-type L1polypeptides. Thus, a series of expression constructions was designed inwhich the artificial EcoRv site was eliminated. The L1 sequence for thisseries of expression constructs was designated HPV 16 L1 Δ C*.

To generate an expression construct containing the HPV 16L1 Δ C*sequence, two PCR reactions were performed to amplify two overlappingfragments from the pUC-HPV16 L1 Δ C encoding E7 amino acids 1-50. Theresulting DNA fragments overlapped at the position of the L1/E7 boundarybut did not contain the two EcoRV restriction sites. Fragment 1 wasgenerated using primers P1 (SEQ ID NO: 21) and P2 (SEQ ID NO: 22) andfragment 2 using primers P3 (SEQ ID NO: 23) and P4 (SEQ ID NO: 24).

Primer P1 SEQ ID NO: 21 GTTATGACATACATACATTCTATG Primer P2 SEQ ID NO: 22CCATGCATTCCTGCTTGTAGTAAAAATTTGCGTCC Primer P3 SEQ ID NO: 23CTACAAGCAGGAATGCATGGAGATACACC Primer P4 SEQ ID NO: 24CATCTGAAGCTTAGTAATGGGCTCTGTCCGGTTCTG

Following the first two amplification reactions, the two purifiedproducts were used as templates in another PCR reaction using primers P1and P4 only. The resulting amplification product was digested withenzymes EcoNI and HindIII inserted into the HPV 16L1 Δ C expressionconstruct described above following digestion with the same enzymes. Theresulting expression construct differed from the original HPV16L1 Δ Cconstruct with DNA encoding L1 and E7 amino acids 1-50 by loss of thetwo internal EcoRV restriction sites. The first EcoRV site was replacedby DNA encoding native L1 alanine and glycine amino acids in thisposition and the second was replaced by a translational stop signal. Inaddition, the expression construct, designated HPV 16 L1 Δ C* E7 1-52,contained the first 52 amino acids of HPV 16 E7 as a result of usingprimer P4 which also encodes E7 amino acids residues histidine atposition 51 and tyrosine at position 52. HPV 16 L1 Δ C* E7 1-52 was thenused to generate additional HPV 16 L1 Δ C expression constructs furtherincluding DNA encoding E7 amino acids 1-55 using primer P1 (SEQ ID NO:21) in combination with primer P5 (SEQ ID NO: 25), E7 amino acids 1-60with primer pair P1 and P6 (SEQ ID NO: 26), and E7 amino acids 1-65 withprimer pair P1 and P7 (SEQ ID NO: 27). The additional aminoacid-encoding DNA sequences in the amplification products arose fromdesign of the primers to include additional nucleotides for the desiredamino acids.

Primer P5 SEQ ID NO: 25 CATCTGAAGCTTAACAATATTGTAATGGGCTCTGTCCG Primer P6SEQ ID NO: 26 CATCTGAAGCTTACTTGCAACAAAAGGTTACAATATTGTAATGGGCTCTG TCCGPrimer P7 SEQ ID NO: 27CATCTGAAGCTTAAAGCGTAGAGTCACACTTGCAACAAAAGGTTACAATA TTGTAATGGGCTCTGTCCGSimilarly, HPV 16 L1 Δ C* E7 1-70 was generated using template DNAencoding HPV 16 L1 Δ C* E7 1-66 and the primer pair P1 and P8 (SEQ IDNO: 28).

Primer P8 SEQ ID NO: 28 CATCTGAAGCTTATTGTACGCACAACCGAAGCGTAGAGTCACACTTGFollowing each PCR reaction, the amplification products were digestedwith EcoNI and HindIII and inserted into HPV16L1 Δ C previously digestedwith the same enzymes. Sequences of each constructs were determinedusing an Applied Biosystems Prism 377 sequencing instrument withfluorescent chain terminating dideoxynucleotides [Prober et al., Science238:336-341 (1987)].

EXAMPLE 2 Generation of Recombinant Baculoviruses

Spodoptera frugiperda (59) cells were grown in suspension or monolayercultures at 27° in TNMFH medium (Sigma) supplemented with 10% fetal calfserum and 2 mM glutamine. For HPV 16 L1-based recombinant baculovirusconstruction, Sf9 cells were transfected with 10 μg of transfer plasmidtogether with 2 μg of linearized Baculo-Gold DNA (PharMingen, San Diego,Calif.). Recombinant viruses were purified by according tomanufacturer's suggested protocol.

To test for expression of HPV 16 L1 protein, 10⁵ Sf9 cells were infectedwith baculovirus recombinant at a multiplicity of infection (m.o.i) of 5to 10. After incubation for three to four days at 28° C., media wasremoved and cells were washed with PBS. The cells were lysed in SDSsample buffer and analyzed by SDS-PAGE and Western blotting usinganti-HPV16 L1 and anti-HPV16 E7 antibodies.

In order to determine which of the chimeric L1 protein expressionconstructs would preferentially produce capsomeres, extracts fromtransfected cells were subjected to gradient centrifugation. Fractionsobtained from the gradient were analyzed for L1 protein content byWestern blotting and for VLP formation by electron microscopy. Theresults are shown in Table 1.

The intact HPV L1 protein, as well as the expression products HPV 16L1Δ310 and HPV 16 L1ΔC, each were shown to produce capsomeres andvirus-like particles in equal proportions. When E7 coding sequences wereinserted into the HPV 16 L1Δ310 vector, only fusion proteins includingE7 amino acids 1 to 50 produced gave rise to detectable capsomereformation.

When E7 encoding DNA was inserted into the HPV 16 L1ΔC vector, allfusion proteins were found to produce capsomeres; chimeric proteinsincluding E7 amino acid residues 40-98 produced the highest level ofexclusively capsomere structures. Chimeric proteins including E7 aminoacids 1-98 and 25-75 both produced predominantly capsomeres, eventhorough virus-like particle formation was also observed. The chimericprotein including E7 amino acids 1-60 resulted in nearly equal levels ofcapsomere and virus-like particle production.

When E7 sequences were inserted into the HPV 16 L1Δ*C vector, all fusionproteins were shown to produce capsomeres. Insertion of DNA encoding E7residues 1-52, 1-55, and 1-60 produced the highest level of capsomeres,but equal levels of virus-like particle production were observed. Whileinsertion of DNA encoding E7 DNA for residues 1-65, 1-70, 25-75, 40-98,and 1-98 resulted in comparatively lower levels or undetectable levelsof capsid, capsomeres were produced in high quantities.

TABLE 1 Capsomeree and Capsid Forming Capacity of Chimeric HPV L1Proteins L1 Expression Capsomere Capsid Construct Insert Yield Yield HVP16 L1 None +++++ +++++ HPV 16 L1 Δ 310 None +++ ++ HPV 16 L1 Δ C None++++ ++++ HPV 16 L1 Δ 310 E7 1-98 − − HPV 16 L1 Δ 310 E7 1-50 ++ − HPV16 L1 Δ 310 E7 25-75 − − HPV 16 L1 Δ 310 E7 50-98 − − HPV 16 L1 Δ C E71-98 +++ + HPV 16 L1 Δ C E7 25-75 +++ + HPV 16 L1 Δ C E7 50-98 + + HPV16 L1 Δ C E7 1-60 +++++ +++++ HPV 16 L1 Δ C E7 40-98 ++++ − HPV 16 L1 ΔC Influenza +++ + HPV 16 L1 Δ *C E7 1-52 +++++ +++++ HPV 16 L1 Δ *C E71-55 +++++ +++++ HPV 16 L1 Δ *C E7 1-60 +++ ++++ HPV 16 L1 Δ *C E7 1-65++ − HPV 16 L1 Δ *C E7 1-70 ++ −

EXAMPLE 3 Purification of Capsomeres

Trichopulsia ni (TN) High Five cells were grown to a density ofapproximately 2×10⁶ cells/ml in Ex-Cell 405 serum-free medium (JRHBiosciences). Approximately 2×10⁸ cells were pelleted by centrifugationat 1000×g for 15 minutes, resuspended in 20 ml of medium, and infectedwith recombinant baculoviruses at m.o.i of 2 to 5 for 1 hour at roomtemperature. After addition of 200 ml medium, cells were plated andincubated for 3 to 4 days at 27° C. Following incubation, cells wereharvested, pelleted, and resuspended in 10 ml of extraction buffer.

The following steps were performed at 4° C. Cells were sonicated for 45seconds at 60 watts and the resulting cell lysate was centrifuged at10,000 rpm in a Sorval SS34 rotor. The supernatant was removed andretained while the resulting pellet was resuspended in 6 ml ofextraction buffer, sonicated for an additional 3 seconds at 60 watts,and centrifuged again. The two supernatants were combined, layered ontoa two-step gradient containing 14 ml of 40% sucrose on top of 8 ml ofCsCl solution (4.6 g CsCl per 8 ml in extraction buffer), andcentrifuged in a Sorval AH629 swinging bucket rotor for 2 hours at27,000 rpm at 10° C. The interface region between the CsCl and thesucrose along with the CsCl complete layer were collected into 13.4 mlQuickseal tubes (Beckman) and extraction buffer added to adjust thevolume 13.4 ml. Samples were centrifuged overnight at 50,000 rpm at 20°C. in a Beckman 70 TI rotor. Gradients were fractionated (1 ml perfraction) by puncturing tubes on top and bottom with a 21-gauge needle.Fractions were collected from each tube and 2.5 μl of each fraction wereanalyzed by a 10% SDS-polyacrylamide gel and Western blotting using ananti-HPV16 L1 antibody.

Virus-like particles and capsomeres were separated from the fractionsidentified above by sedimentation on 10 to 50% sucrose gradients. Peakfractions from CsCl gradients were pooled and dialyzed for 2 hoursagainst 5 mM HEPES (pH 7.5). Half of the dialysate was used to producecapsomeres by disassembly of intact VLPs overnight by adding EDTA (finalconcentration 50 mM), EGTA (50 mM), DTT (30 mM). NaCl (100 mM), andTris/HCl, pH 8.0, (10 mM). As control, NaCl and Tris/HCl only were addedto the other half.

For analysis of capsomeres produced from disassembled VLPs, EDTA, EGTA,and DTT (final concentration 5 mM each) were added to the sucrosecushions which were centrifuged at 250,000×g for 2 to 4 hours at 4° C.Fractions were collected by puncturing tubes from the bottom. A 1:10dilution of each fraction was then analyzed by antigen capture ELISA.

EXAMPLE 4 Immunization Protocol for Production of Polyclonal Antiseraand Monoclonal Antibodies

Balb/c mice are immunized subcutaneously three times, every four weekswith approximately 60 μg of HPV chimeric capsomeres mixed 1:1 withcomplete or incomplete Freund's Adjuvants in a total volume of 100 μl.Six weeks after the third immunization, mice are sacrificed and blood iscollected by cardiac puncture.

EXAMPLE 5 Peptide ELISA to Quantitate Capsomere Formation

Microtiter plates (Dynatech) are coated overnight with 50 μl of peptideE701 [Muller et al., 1982] at a concentration of 10 μg/ml in PBS. Wellsare blocked for 2 hour at 37° C. with 100 μl of buffer containing 5% BSAand 0.05% Tween 20 in PBS and washed three times with PBS containing0.05% Tween 20. After the third wash, 50 μl of sera diluted 1:5000 inBSA/Tween 20/PBS is added to each well and incubation carried out for 1hour. Plates are washed again as before and 50 μl of goat-anti-mouseperoxidase conjugate is added at a 1:5000 dilution. After 1 hour, platesare washed and stained using ABTS substrate (0.2 mg/ml,2,2′-Azino-bis(3-ethylbenzhiazoline-β-sulfonic acid in 0.1 MNa-Acetate-Phosphate buffer (pH 4.2) with 4 μl 30% H₂O₂ per 10 ml).Extinction is measured after 1 hour at 490 nm in a Dynatech automatedplate reader.

EXAMPLE 6 Antigen Capture ELISA to Quantitate Capsomere Formation

To allow relative quantification of virus-like particles and capsomeresin fractions of CsCl gradients, an antigen capture ELISA was utilized.Microtiter plates were coated overnight with 50 μl/well of a 1:500dilution (final concentration of 2 μg per ml, in PBS) with a protein Apurified mouse monoclonal antibody immunospecific for HPV 16 L1(antibodies 25/C, MM07 and Ritti 1 were obtained from mice immunizedwith HPV 16 VLPs). Plates were blocked with 5% milk/PBS for 1 hour and50 μl of fractions of CsCl gradients were added for 1 hour at 37° C.using a 1:300 dilution (in 5% milk/PBS). After three washings withPBS/0.05% Tween 20, 50 μl of a polyclonal rabbit antiserum (1:3000dilution in milk/PBS), raised against HPV 16 VLPs was added and plateswere incubated at 37° for 1 hour. Plates were washed again and furtherincubated with 50 μl of a goat-anti-rabbit peroxidase conjugate (Sigma)diluted 1:5000 in PBS containing 5% milk for 1 hour. After finalwashing, plates were stained with ABTS substrate for 30 minutes andextinction measured at 490 nm in a Dynatech automated plate reader. As anegative control, the assay also included wells coated only with PBS.

To test monoclonal antibodies for capsomere specificity, VLPs withEDTA/DTT to disassemble particles. Treated particle preparations wereassayed in the antigen-capture ELISA and readings compared to untreatedcontrols. For disassembly, 40 μl of VLPs was incubated overnight at 4°C. in 500 μl of disruption buffer containing 30 mM DTT, 50 mM EGTA, 60mM EDTA, 100 mM NaCl, and 100 mM Tris/HCl, pH 8.0. Aliquots of treatedand untreated particles were used in the above capture ELISA in a1:20-1:40 dilution.

EXAMPLE 7 Hemagglutinin Inhibition Assay

In order to determine the extent to which chimeric capsomere vaccinesevoke production of neutralizing antibodies, a hemagglutinationinhibition assay is carried out as briefly described below. This assayis based on previous observations that virus-like particles are capableof hemagglutinizing red blood cells.

Mice are immunized with any of a chimeric capsomere vaccine and sera iscollected as described above in Example 4. As positive controls, HPV16L1 virus like particles (VLPs) and bovine PV1 (BPV) L1 VLPs are assayedin parallel with a chimeric capsomere preparation. To establish apositive baseline, the HPV16 or BPV1 VLPs are first incubated with orwithout sera collected from immunized mice after which red blood cellsare added. The extent to which preincubation with mouse cera inhibitsred blood cell hemagglutinization is an indication of the neutralizingcapacity of the mouse sera. The experiments are then repeated usingchimeric capsomeres in order to determine the neutralizing effect of themouse sera on the vaccine. A brief protocol for the hemagglutinationinhibition assay is described below.

One hundred microliters of heparin (1000 usp units/ml) are added to 1 mlfresh mouse blood. Red blood cells are washed three times with PBSfollowed by centrifugation and resuspension in a volume of 10 ml. Next,erythrocytes are resuspended in 0.5 ml PBS and stored at 4° C. for up tothree days. For the hemagglutinin assay, 70 μl of the suspension is usedper well on a 96-well plate.

Chimeric capsomere aliquots from CsCl gradients are dialyzed for onehour against 10 mM Hepes (pH 7.5) and 100 μl of two-fold serialdilutions in PBS are added to mouse erythrocytes in round-bottom 96-wellmicrotiter plates which are further incubated for 3-16 hours at 4° C.For hemagglutination inhibition, capsomeres are incubated with dilutionsof antibodies in PBS for 60 minutes at room temperature and then addedto the erythrocytes. The level of erythrocyte hemagglutination, andtherefore the presence of neutralizing antibodies, is determined bystandard methods.

In preliminary results, mouse sera generated against chimeric capsomerescomprising HPV16L1ΔC protein in association with E7 amino acid residues1-98 was observed to inhibit hemagglutination by HPV16 VLPs, but not byBPV VLPs. The mouse sera was therefore positive for neutralizingantibodies against the human VLPs and this differential neutralizationwas most likely the result of antibody specificity for epitopes againstwhich the antibodies were raised.

Numerous modifications and variations in the invention as set forth inthe above illustrative examples are expected to occur to those skilledin the art. Consequently only such limitations as appear in the appendedclaims should be placed on the invention.

1. A method for producing a vaccine formulation comprising a humanpapilloma virus capsomere, said capsomere comprising a fusion proteincomprising a human papilloma virus L1 protein adjacent amino acidresidues from a second protein, said method comprising the steps ofexpressing and purifying human papilloma virus L1 proteins.
 2. A methodfor producing a vaccine formulation comprising a human papilloma viruscapsomere, said capsomere comprising a truncated human papilloma virusL1 protein having a deletion of one or more amino acid residuesnecessary for formation of a virus-like particle, said method comprisingthe steps of expressing and purifying truncated human papilloma virus L1proteins.
 3. The method of claim 2 wherein said capsomere furthercomprises a fusion protein comprising amino acid residues from a secondprotein adjacent to said truncated human papilloma virus L1 protein. 4.The method of any one of claims 1, 2, or 3 wherein the L1 protein isencoded in the genome of a human papilloma virus selected from the groupconsisting of HPV6, HPV11, HPV 16, HPV 18, HPV33, HPV35, and HPV45. 5.The method of claim 4 wherein the papilloma virus is HVP16.
 6. Themethod of any one of claims 2, 3, or 5 wherein carboxy terminal aminoacid residues are deleted from the L1 protein.
 7. The method of claim 6wherein 1 to 34 carboxy terminal amino acid residues are deleted fromthe L1 protein.
 8. The method of claim 7 wherein 34 carboxy terminalamino acid residues are deleted from the L1 protein.
 9. The method ofany one of claims 2, 3, or 5 wherein amino terminal amino acid residuesare deleted from the L1 protein.
 10. The method of any one of claims 2,3, or 5 wherein internal amino acid residues are deleted from the L1protein.
 11. The method of claim 10 wherein the amino acid residuesdeleted from the L1 protein comprise a nuclear localization signal. 12.The method of claims 2 or 3 wherein the amino acids residues from thesecond protein are derived from an HPV protein.
 13. The method of claim12 wherein the HPV protein is an early HPV protein.
 14. The method ofclaim 12 wherein the early HPV protein is selected from the groupconsisting of E1, E2, E3, E4, E5, E6, and E7.